Toxicity of Beauveria bassiana to Bactrocera dorsalis and effects on its natural predators

Entomopathogenic fungi (EPF) are economical and environmentally friendly, forming an essential part of integrated pest management strategies. We screened six strains of Beauveria bassiana (B1–B6) (Hypocreales: Cordycipitaceae), of which B4 was the most virulent to Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). We further assessed the biological characteristics of strain B4 and the environmental factors influencing its ability to infect B. dorsalis. We also evaluated the effects of B4 on two of the natural predators of B. dorsalis. We found that strain B4 was the most virulent to 3rd instar larvae, pupae, and adult B. dorsalis, causing mortality rates of 52.67, 61.33, and 90.67%, respectively. B4 was not toxic to B. dorsalis eggs. The optimum B4 effects on B. dorsalis were achieved at a relative humidity of 91–100% and a temperature of 25°C. Among the six insecticides commonly used for B. dorsalis control, 1.8% abamectin emulsifiable concentrate had the strongest inhibitory effect on B4 strain germination. B4 spraying affected both natural enemies (Amblyseius cucumeris and Anastatus japonicus), reducing the number of A. cucumeris and killing A. japonicus adults. We found a valuable strain of EPF (B4) that is virulent against many life stages of B. dorsalis and has great potential for the biological control of B. dorsalis. We also provide an important theoretical and practical base for developing a potential fungicide to control B. dorsalis.


Introduction
Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is one of the most economically serious invasive pests worldwide (Vontas et al., 2011).It is widely distributed in tropical and subtropical regions, damaging a wide variety of fruits and fleshy vegetables (Liu et al., 2019;Li et al., 2021).This fruit fly mainly lays eggs inside fruits and vegetables as an adult, and the larvae subsequently feed on the fruits and cause damage.It is precisely this concealed method of damage by fruit flies that makes their prevention and control challenging (Wakil et al., 2022).To combat the harmful fruit fly, growers largely rely on chemical insecticides.However, although effective, they have many adverse effects with long-term use, including resistance problems, non-target effects, and harm to humans and the environment (Jin et al., 2011a;Hsu et al., 2012;Khan et al., 2014;Chen et al., 2019;Awan et al., 2021).
Due to the range of issues associated with the use of chemical insecticides, it is essential to develop more environmentally friendly fruit fly control methods (Daane and Johnson, Li et al. 10.3389/fmicb.2024.1362089Frontiers in Microbiology 02 frontiersin.org2010).Biological control methods based on natural predators, parasitoids, and entomopathogenic fungi (EPF) are regarded as safe substitutes for chemical pesticides (Dorta et al., 2020).For a long time, exploring the use of pathogenic microorganisms as a strategy against pests has been a focal point of research (Hajek and St Leger, 1994).
We selected the B4 strain, which was the most virulent to all life stages of B. dorsalis, from the six B. bassiana strains, and evaluated the effects of environmental factors (temperature, relative humidity, and relative soil water content), different concentrations, and chemical reagents on the virulence of B. bassiana (B4) on B. dorsalis and two of its natural enemies.Our research provides an important theoretical and practical basis for developing targeted B. bassiana agents and insecticide applications to effectively control different life stages of B. dorsalis in the field.

Beauveria bassiana strain cultures
We obtained six strains of B. bassiana (B1-B6).Strain B1 was provided by the Laboratory of Fungal Pesticide Creation, Institute of Agricultural Environment and Sustainable Development, Chinese Academy of Agricultural Sciences (Beijing, China).Strains B2-B4 were provided by the Biocontrol Laboratory, Institute of Plant Protection, Guangdong Academy of Agricultural Sciences (Guangzhou, China).Strain B5 was provided by the Insect Ecology Laboratory, South China Agricultural University (Guangzhou, China).Strain B6 was collected from B. dorsalis in a starfruit plantation (Boluo County,Guangdong Province,China;23°23′N,114°30′E).
Beauveria bassiana strains were incubated on Sabouraud dextrose agar with a 1% yeast (SDAY) medium at 25°C for 7-10 days, with a 16:8-h (light:dark) regime (Pan et al., 2008;Zhang et al., 2010).Conidia were isolated using the method of Ekesi et al. (2003), after which the mature conidia were diluted using 0.1% Tween-80 in sterile water to prepare a suspension with a concentration of 1.0 × 10 8 conidia/mL.A solution of 0.1% Tween-80 in sterile water was used as the control.

Determining Beauveria bassiana virulence
To accurately determine the virulence of the six strains of B. bassiana, the 1.0 × 10 8 concentration was used to determine its pathogenicity to B. dorsalis (Pan et al., 2008).Eggs, 3rd instar larvae, and pupae of B. dorsalis were dipped in a spore suspension of 1.0 × 10 8 conidia/mL for 30 s (Pan et al., 2008).The eggs were then placed in a rearing box with artificial feed.We used 3rd instar larvae because, in the field, the first two larval instars feed within the host plant; after reaching the third instar, they emerge from the host plant and drop to the soil to pupate (Susanto et al., 2022).At this point, they may come into contact with other materials and substrates, including soil containing B. bassiana.Therefore, only 3rd instar larvae were tested.
The 3rd instar larvae and pupae were transferred to plastic boxes containing moist sandy soil (water mixed with sandy soil, keeping the surface of the sandy soil in contact with water, but not saturating it, to avoid particles sticking together) and reared at 25°C.Egg hatching, larval pupation, and pupal emergence was recorded daily.Adults were treated using the film method (Lohse et al., 2015).Specifically, we used a hand-held sprayer to evenly spray an equal volume (5 mL) of the suspension containing 1.0 × 10 8 conidia/mL on the internal wall of a 250-mL triangular bottle.The suspension was air dried.We collected an initial 30 emerging adults over 1 to 2 days, while adding feed and water, and observed the mortality rate over 15 days.Thirty eggs (freshly laid eggs, 1-2 h old), 3rd instar larvae, pupae (1-2 days after pupation), and adults were used in each treatment (six different B. bassiana strains for each B. dorsalis life stage), with five replicates of observations starting from the third day of the treatment (observations were made up to day 15, five times in total).

Effect of environmental factors on the virulence of the B4 strain
The effect of temperature on the virulence of the B4 strain was tested at eight different temperatures (13, 16, 19, 22, 25, 28, 31, and 34°C, observed for 10 days).The flies were reared at 95% relative humidity and 16:8-h light:dark cycle in an artificial climatic chamber to observe the effect of temperature on mortality.The treatment of the different B. dorsalis life stages with strain B4 followed the procedures outlined in Section 2.4.
The effect of RH on the virulence of the B4 strain involved exposing different B. dorsalis life stages to a constant relative humidity in four different ranges, i.e., 60-70, 71-80, 81-90, and 91-100% RH.The treatment of the different B. dorsalis life stages with strain B4 followed the procedures outlined in Section 2.4.The larvae, pupae, and adults were observed for 10 days at 25°C after inoculation.
The combined effect of temperature and RH on B4 strain growth was also assessed.Discs were collected from the edge of B4 colonies that had been cultured for 7 days using a 5-mm diameter paper punch.The discs were inoculated into the center of SDAY medium, marked with two points at the bottom of the Petri dish to highlight the original diameter, and incubated under different temperature and RH conditions.Subsequently, the diameter of the colonies was measured every 2 days.The final data was recorded on the 10th day (with five replicates).
To determine the effect of the relative soil moisture content on the virulence of the B4 strain, we used loose sandy soil with a field water holding capacity of 4.5%.Before test initiation, the soil was sieved to remove impurities and then baked in an oven at 105°C for 5-6 h until it reached a constant mass.Six different relative soil water levels (40, 50, 60, 70, 80, and 90%) were obtained following the protocol of Hou et al. (2022).We sprayed 5 mL of a 1.0 × 10 7 conidia/mL suspension evenly on the soil surface at different RHs with a hand-held sprayer.The 3rd instar larvae were then placed on the soil and the number of larvae pupating was recorded daily (six treatments, 30 larvae per treatment, five replicates).For the pupal virulence test, pupae that had pupated 1-2 days prior were placed on soil with different relative water contents, a hand-held sprayer was used to apply 5 mL of a suspension of 1.0 × 10 7 conidia/mL to the soil surface, and the number of emerging adults was recorded daily (six treatments, 30 pupae per treatment, five replicates).The relative water content of the test soils was maintained by weighing the samples at 12 h intervals at 25°C and supplementing them with water to account for any evaporatory loss to maintain a consistent water content.

Effect of common chemical agents on the biological characteristics of the B4 strain
The six insecticides were tested at three different concentrations.The first concentration involved using the recommended field concentration (concentrations recommended for routine use in the instructions), which is considered a lethal dose.The second concentration was the conventionally used concentration diluted five times, considered a sublethal dose, whereas the third concentration was the conventionally used concentration diluted ten times, considered a low sublethal dose (Xu et al., 2002).
The B4 strain, cultured for 10 days (25°C, 16:8-h light:dark), was used to prepare a conidial suspension using sterile water containing 0.1% Tween-80.Each agent was adjusted to the desired concentration using 1 mL of B4 conidia suspension containing 5% sucrose.We transferred 100 μL of the solution to a sterile slide and placed it in a Petri dish lined with filter paper, added sterile water dropwise to the dish to maintain 100% RH, and incubated it for 24 h to observe spore germination using a microscope (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany; in five replications).
We applied 0.1 mL of the insecticide solution to evenly coat the surface of the SDAY medium.We transferred a 5-mm diameter disc of the B4 strain (cultured at 25°C and 95% RH for 7 days) to the center of the medium containing the insecticide.The sampling method of the disc followed the procedure described in Section 2.6.We marked two points at the bottom of the Petri dish to highlight the initial diameter; each treatment was replicated five times.The colony diameter was measured on the 5th and 10th d.We used the culture medium without pesticide as the control.

Effect of strain B4 on the natural enemies of Bactrocera dorsalis
The effect of strain B4 on A. cucumeris was assessed.The concentration of spore powder in 0.1 g was calculated to be 7.0 × 10 9 conidia/g using haemocytometry (Mulatu et al., 2021).We created three treatments of B. bassiana with 0.5, 2.5, and 12.5 g (based on pre-experimental results and the characteristics of the feeding patterns of A. cucumeris) (Zhang et al., 2002;Li, 2017).We mixed the samples with 500 g of bran and 10 g of seeds containing approximately 300 mites (in five replicates).The mite population was observed on the 10th and 20th days after inoculation.Five samples (of 0.1 g each) were randomly collected and observed using a dissecting microscope.
The effect of strain B4 on A. japonicus involved different concentrations (based on the results of the previous experiments and the characteristics of the feeding patterns of A. japonicus) (Zhao et al., 2023).Spore suspensions of 1.0 × 10 5 , 1.0 × 10 6 , 1.0 × 10 7 , 1.0 × 10 8 , and 1.0 × 10 9 conidia/mL were evenly sprayed on the wall of a tube (sterilized glass tubes with a diameter of 15 cm and a height of 20 cm) and air dried.Each tube was inoculated with 50 A. japonicus at 1-2 days after emergence and 50 A. pernyi eggs.Parasitic holes in the eggs were evaluated every 5 days, and the experiment was terminated on day 20.Parasitized eggs were recorded for each treatment.

Statistical analysis
Calculations of LC 50 and LT 50 with corresponding 95% confidence limits (CL) were first corrected for mortality and then subjected to probit regression analyses using the SPSS v.22.0 software (SPSS Inc., Chicago, IL).The SAS software (version 9.4) was used for the other data analyses.The experimental results were analyzed using Duncan's multiple range test (DMRT), with p < 0.05 considered statistically significant.Normal distribution was verified before data analysis, followed by ANOVA.The calculation of the corrected mortality was as follows:

Virulence of different strains of Beauveria bassiana against Bactrocera dorsalis
The six strains of B. bassiana were not highly virulent to B. dorsalis eggs (Table 1).For 3rd instar larvae, pupae, and adults, the virulence of each strain increased with increasing treatment duration.Strain B4 had the highest virulence in every observation, reaching 52.67% (3rd instar larvae), 61.33% (pupae), and 90.67% (adults) at day 15, which was significantly higher than that of all the other strains (Table 1).Therefore, we focused entirely on strain B4 in all subsequent experiments.
At the same concentration (except for 1.0 × 10 3 conidia/mL), there were some differences in the LT 50 between the larvae, pupae, and adults, suggesting that tolerance to B4 varies between the three life stages.The LT 50 of each B. dorsalis stage showed a decreasing trend with increasing B4 strain concentration (Table 2).Strain B4 was most toxic to adults, and the LT 50 was less than 10 days at concentrations as low as 1.0 × 10 6 conidia/mL.
The mortality of B. dorsalis between the three relative soil water contents of 50, 60, and 70% did not vary significantly.The variations in the virulence of the B4 strain between the different water contents (40, 80, and 90%) were highly significant.In addition, the highest mortality rate was 79.21% for larvae at a relative soil moisture content of 60 and 80.63% for pupae at a relative soil moisture content of 50% (Table 7).

Common insecticide effects on the biological characteristics of the B4 strain
At three different doses, 1.8% avermectin EC had the strongest inhibitory effect on the germination of B4 conidia, and conidia were unable to germinate at the lethal dose (Table 8).The remaining five agents showed weak inhibition of conidial germination at both lethal and sublethal doses, but were not significantly different from the control at the low sublethal dose (Table 8).
Similarly, 1.8% avermectin EC had the strongest inhibitory effect on the growth of B4 strains at lethal and sublethal concentrations, followed by 48% Lorsban EC (Table 9).While 1.8% abamectin EC still had a strong inhibitory effect on B4 strain growth at sublethal doses, there was no difference between the other five agents.Therefore, the effect of 1.8% abamectin EC on the growth of the B4 strain at low sublethal concentrations was further measured.It was found that the inhibitory effect was still strong (57.14% on day 5 and 71.05% on day 10).This indicates that avermectin cannot be used in conjunction with B4.

Effect of the B4 strain on natural enemies of Bactrocera dorsalis
The B4 strain significantly inhibited A. cucumeris reproduction, and the inhibitory effect increased with increasing treatment concentrations.Extending the treatment time weakened the inhibitory effect of the B4 strain on the mites (Table 10).For A. cucumeris, increasing both the treatment time and concentration significantly increased the mortality, especially at high concentrations   of 1.0 × 10 9 conidia/mL.More than half the mites died by day 15 (52%) (Table 11).
The A. japonicus oviposition in the control was higher than that in the B4-treated samples at the same time, while higher treatment concentrations resulted in greater variation in A. japonicus than in the control (Table 11).

Discussion
Currently, in the field of biological control, EPF are used to control insect pests and have the potential to free producers from a heavy      dependence on chemical pesticides (Legaspi et al., 2000;Lovett and Leger, 2017).Based on B. bassiana, as one of the most effective EPFs, many commercial products for biological control have been developed (Zimmermann, 2007).In the USSR, B. bassiana products were mainly used to control Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) and Cydia pomonella (Lepidoptera: Tortricidae) (Ferron, 1981).In China, B. bassiana is widely used to control Ostrinia nubilalis (Lepidoptera: Crambidae) in corn and Dendrolimus punctatus (Lepidoptera: Lasiocampidae) on pines (Hussey and Tinsley, 1981).In addition, B. bassiana has been shown to have some pathogenicity in the laboratory against several target and non-target organisms, such as Alphitobius diaperinus (Coleoptera: Tenebrionidae) (Rohrlich et al., 2018) and Chrysoperla externa (Neuroptera: Chrysopidae) (Amorim et al., 2005).The B4 strains screened in this study showed high virulence against each developmental stage of B. dorsalis and have potential for field application in the future biological control of B. dorsalis.The B4 strain caused the highest mortality to adults, followed by pupae and larvae, while eggs were largely unsusceptible.Similar results have been reported for Bactrocera zonata (Mahmoud, 2009;Hussein et al., 2018;Usman et al., 2021) and B. cucurbitae (Hamzah et al., 2021).However, the virulence of larvae and pupae to B. bassiana in previous studies differed from that described here.In B. zonata (Gul et al., 2015;Hussein et al., 2018;Usman et al., 2021) and C. capitata (Soliman et al., 2020) larvae were more susceptible than pupae.Moreover, Usman et al. ( 2021) and Wakil et al. (2022) found that B. dorsalis pupae were more susceptible than larvae, which does not align with our current findings.The contrasting results may be due to differences between the strains of B. bassiana, the method of application, and the age of the pupae (Rizvi et al., 2009;Beris et al., 2013;Gul et al., 2015;Soliman et al., 2020;Shaurub, 2022).We focused on one-to two-day-old pupae, whereas previous research showed that the older the pupae, the less susceptible they are to EPF (Poprawski et al., 1985;Hussein et al., 2018).This may be related to the softer cuticle of young pupae, which allows the fungus to penetrate the epidermis more easily (Mora et al., 2018).
Temperature and humidity are the most important environmental factors affecting the growth and virulence of B. bassiana (Dorschner et al., 1991;Shaurub, 2022).Our results are consistent with Pan et al. (2008), who found that 25°C and 90-100% RH were the optimum temperature and humidity for spore germination and colony growth.The virulence of B. bassiana to each stage of B. dorsalis was optimized under these conditions.The soil moisture content is critical for the larval pupation and pupal emergence (Ekesi et al., 2003;Quesada-Moraga et al., 2006).The virulence of B. bassiana is the highest at a soil water content of 50-70%, and B. bassiana may grow best under these conditions.The growth and infestation of B. bassiana are influenced by various environmental factors.Therefore, spraying chemicals in cloudy weather or after rain is conducive to B. bassiana germinating to achieve optimal control effects (Pan et al., 2014).
Combining chemical agents with EPF is a promising pest control option to minimize harmful chemical effects (Karthiba et al., 2010;Pelizza et al., 2018).Mixing B. bassiana and deltamethrin was effective in controlling Hyalomma anatolicum (Acari: Ixodidae) (Sun et al., 2011).The inhibitory effect of beta cypermethrin on spore formation and conidial germination of B. bassiana was the lowest at the recommended concentration of 10%, and the mortality rate of Phauda flammans (Lepidoptera: Phaudidae) was significantly increased when B. bassiana was mixed with beta cypermethrin at the recommended concentration of 10% (Chen et al., 2021).However, in the current study, 1.8% avermectin EC showed high inhibition of the B4 strain, while the other agents had a relatively small effect.This indicates that 1.8% avermectin EC should not be mixed with the B4 strain or applied  The combined use of EPF and arthropod natural enemies in an integrated pest management strategy has been previously explored (González-Mas et al., 2019).Several studies have examined the safety and efficacy of combining EPF and other biocontrol components, e.g., predators, parasitoids, and nematodes (Roy and Pell, 2000;Acevedo et al., 2007;Labbé et al., 2009;Ansari et al., 2010;Martins et al., 2014).Gadhave et al. (2016) indicated that B. bassiana does not affect the parasitoid species associated with pea leafminer.Similar results were obtained for Myzus persicae (Hemiptera: Aphididae) and its parasite Aphidius colemani (Hymenoptera: Braconidae) (Jaber and Araj, 2018).However, we found that the B4 strain had a constraining effect on the growth, development, and reproduction of A. cucumeris and A. japonicus populations.This is consistent with the findings by Gao et al. (2022) that B. bassiana was highly pathogenic to non-target Spodoptera frugiperda (Lepidoptera: Noctuidae).The host range of B. bassiana is very wide, so consideration of its effects on non-target species is an important factor when using EPF in integrated pest management (Rohrlich et al., 2018).Therefore, it is important to avoid releasing A. cucumeris and A. japonicus at the same time as applying B. bassiana in the field.Furthermore, the relatively low diversity within agroecosystems may influence natural enemies within (Schmitz, 2007;Cappa et al., 2022).Field application of the B4 strain should therefore consider not only the natural enemies of B. dorsalis, but also the protection of the natural enemies of other pest species to prevent significant losses within agroecosystems, indirectly exacerbating the environmental pollution caused by chemical pesticides.

Conclusion
B4 controls several B. dorsalis life stages, particularly under optimal environmental conditions for growth and virulence.We also provide an empirical basis for combining the B4 strain with chemicals and natural enemies of B. dorsalis for improved virulence in the field.In summary, we provide an important practical base for the development of B. bassiana formulations to aid in the control of B. dorsalis in the field.The author(s) declare that financial support was received for the research, authorship, and/or publication of this article.This research was supported by the National Key R&D Project of China (2021YFC2600404) to Y-xQ, Guangdong Modern Agriculture The calculation of the growth inhibition rate of B. bassiana colonies was as follows: same number and letter in the same column indicate no significant variation between them in the DMRT test (p ≥ 0.05).

TABLE 1
Virulence of different B. bassiana strains against B. dorsalis at the concentration of 1.0 × 10 8 conidia/mL.
e Data with the same number and letter in the same column indicate no significant difference in the Duncan's Multiple Range Test (DMRT) test (p ≥ 0.05).

TABLE 2
LT 50 of the B4 strain at different concentrations in B. dorsalis.Data with the same number and letter in the same column are not significantly different using the DMRT test (p ≥ 0.05).

TABLE 3
Cumulative mortality and toxicity of the B4 strain against B. dorsalis in different temperatures.
CL, confidence limits; R, correlation index; Data with the same number and letter in the same column have P ≥ 0.05 (DMRT test); *Significant difference (P ≤ 0.05).

TABLE 4
Effect of temperature on the growth of the B4 strain.Data with the same number and letter in the same column indicate no significant difference in the DMRT test (p ≥ 0.05).

TABLE 5
Cumulative mortality and toxicity of the B4 strain against B. dorsalis with various humidity.
CL, confidence limits; R, correlation index; Data with the same number and letter in the same column indicate no significant variation in the DMRT test (p ≥ 0.05); *Significantly different (P ≤ 0.05).

TABLE 6
Effect of humidity on the growth of the B4 strain.

TABLE 7
Effect of the relative soil water content on the virulence of the B4 strain.Data with the same number and letter in the same column indicate no significant variation between them by the DMRT test (p ≥ 0.05).

TABLE 8
Effects of different dilution ratios of chemical reagents on the conidial germination rate of the B4 strain.Data with the same number and letter in the same column indicate no significant variation between them by the DMRT test (p ≥ 0.05).

TABLE 9
Growth inhibition rate of the B4 strain using various chemical agent doses.Data with the same number and letter in the same column indicate no significant variation between them in the DMRT test (p ≥ 0.05).

TABLE 10
Lethal effect of the B4 strain on A. cucumeris.

TABLE 11
Effect of the B4 strain on the cumulative mortality and fecundity of A. japonicus.Data with the same number and letter in the same column indicate no significant variation between them in the DMRT test (p ≥ 0.05).